Process for the biological production of 1,3-propanediol from glycerol with high yield

ABSTRACT

The present invention provides a method for the anaerobic production of 1,3 propanediol, by culturing a  Clostridium  strain in an appropriate culture medium comprising glycerol as a source of carbon, wherein said  Clostridium  strain does not produce substantially other products of the glycerol metabolism selected among the group consisting of: butyrate, lactate, butanol and ethanol, and recovering of 1,3-propanediol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage filing of InternationalApplication Serial No. PCT/EP2006/067987 filed Oct. 31, 2006, which isincorporated herein by reference in its entirety.

FIELD OF INVENTION

The invention comprises a process for the bioconversion of glycerol to1,3-propanediol at high yield by a metabolically engineered Clostridium.

BACKGROUND OF THE INVENTION

1,3-propanediol is a monomer use in the production of polyester fibersand with potential use in the manufacture of polyurethanes and cycliccompounds.

1,3-propanediol can be produced by different chemical routes from i)acrolein water and hydrogen ii) ethylene oxide carbon monoxide and waterin the presence of phosphine and from glycerol and hydrogen in thepresence of carbon monoxide. All these methods have in common to beexpensive and to generate waste streams containing polluting substances.

1,3-propanediol can be produced as anacetate/butyrate/lactate/1,3-propanediol mixture by the fermentation ofglycerol by different Clostridia. The general metabolism of glycerolinto Clostridia is presented in FIG. 1.

In one way, glycerol is converted to 1,3-propanediol in a two stepenzymatic reaction sequence. In a first step a glycerol dehydratasecatalyze the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA)and water. In the second step 3-HPA is reduced to 1,3-propanediol by aNADH dependent 1,3-propanediol dehydrogenase. Most of the1,3-propanediol producing clostridia use a B12 dependent glyceroldehydratase encoded by the dhaB1B2B3 structural genes while Clostridumbutyricum uses a B12 independent enzyme encoded by the dhaB1 structuralgene. For the B12 dependent glycerol dehydratases, orfX and orfZ encodethe glycerol dehydratase reactivation factor while for the only knownB12 independent enzyme, dhaB2 encodes an S-Adenosyl-Méthionine (SAM)dependent activation factor. Near the genes encoding the structural andactivation factors a gene encoding a 1,3-propanediol dehydrogenase(dhaT) is also present. Production of 1,3-propanediol from glycerolconsumes NADH.

In another way, when glycerol is not transformed into 1,3-propanediol,it is oxidized to dihydrohycetone-phosphate (DHAP) with the concomitantproduction of NADH by either a glycerol kinase and aglycerol-3-Phosphate dehydrogenase encoded respectively by glpk and glpAor by a glycerol dehydrogenase followed by a DHA kinase encodedrespectively by dhaD and dhaK1K2. DHAP will then enter the glycoliticpathway with the production of pyruvate and acetyl-CoA as keyintermediates. Pyruvate and acetyl-CoA can be reduced to respectivelylactate and ethanol by a lactate dehydrogenase encoded by the ldh geneand a bi-functional aldehyde-alcohol dehydrogenases encoded by adhE.Acetyl-CoA can also be converted to butyryl-CoA, an intermediate productthat can be:

-   -   i) converted to butyric acid by a phospho-transbutyrylase and a        butyrate kinase encoded respectively by the ptb and buk genes or    -   ii) reduced to butanol by a bi-functional aldehyde-alcohol        dehydrogenase encoded by adhE.

In solventogenic clostridia, acetone is produced from aceto-acetyl-CoA(an intermediate in the production of butyryl-CoA) by a CoA-transferaseand an acetoacetate decarboxylase encoded respectively by the ctfAB andadc genes. Hydrogen is produced by an iron only hydrogenase encoded bythe hydA gene.

Both natural and recombinant clostridia produce 1,3-propanediol at amaximal yield of 0.55 g/g of glycerol due to the co-production ofreduced compounds like butyric acid (butyrate), lactic acid (lactate),ethanol or butanol. To increase the yield of 1,3-propanediol productionit is necessary to avoid the production of all the reduced co-productsand associate the production of 1,3-propanediol to an oxidizedco-product.

Clostridium acetobutylicum strains unable to produce butyrate havealready been described in the article (Green et al., 1996). The butyrateformation was dramatically reduced because of the inactivation of thebuk gene obtained by single crossing-over with a non-replicable plasmid.This mutant strain was tested for the production of 1,3-propanediol asshown in (Gonzalez-Pajuelo, 2005, Metabolic Engineering). Thisrecombinant strain effectively produces 1,3-propanediol as the mainfermentation product, but produces also butanol, which decreases the1,3-propanediol yield.

The 1,3-propanediol fermentation of glycerol by Clostridia can run inbatch, fedbatch or continuous cultures.

The problem to be solved by the present invention is the biologicalproduction of 1,3 propanediol from glycerol at high yield, with noconcomitant production of reduced compounds such as butyrate, lactate,or alcohols. This production is performed by anaerobic fermentation withClostridia.

SUMMARY OF THE INVENTION

Applicants have solved the stated problem and the present inventionprovides a method for the anaerobic production of 1,3 propanediol, byculturing a Clostridium strain in an appropriate culture mediumcomprising glycerol as a source of carbon, wherein said Clostridiumstrain does not produce substantially other products of the glycerolmetabolism selected among the group consisting of: butyrate, lactate,butanol and ethanol, and recovering 1,3-propanediol.

The 1,3-propanediol may be produced concomitantly with a single oxidizedproduct of the glycerol metabolism.

In a particular aspect of the invention, the Clostridium strain ismodified to limit production of metabolites from glycerol, whichbiosynthesis pathway is NADH or NADPH consuming, except for1,3-propanediol.

In one aspect of this invention, a Clostridium naturally producing1,3-propanediol is genetically modified to produce 1,3-propanediol athigher yield by deleting:

-   -   i) the gene coding for the butyrate kinase (buk) or the        phospho-transbutyrylase (ptb) to avoid butyrate production    -   ii) optionally, all the genes coding for the lactate        dehydrogenases (ldh) to avoid lactate production    -   iii) optionally, the genes coding for the bi-functional        aldehyde-alcohol dehydrogenases (adhE) to avoid alcohols        formation.

In another aspect of this invention, a Clostridium naturally producingbutyrate but unable to produce 1,3-propanediol is genetically modifiedto produce 1,3-propanediol at high yield. This result is achieved byreplacing the ptb or the buk genes coding for enzymes involved in thebutyrate pathway with the operon of C. butyricum coding for enzymesinvolved in the B12 independent 1,3-propanediol pathway, and bydeleting:

-   -   i) optionally, all the genes coding for the lactate        dehydrogenases (ldh) to avoid lactate production    -   ii) optionally, the genes coding for the bi-functionnal        aldehyde-alcohol dehydrogenases (adhE) to avoid alcohols        formation.

In a further aspect of this invention, a Clostridum naturally producingethanol but unable to produce 1,3-propanediol is genetically modified toproduce 1,3-propanediol. This result is achieved by replacing one of theadhE genes coding for enzymes involved in the ethanol pathway, with theoperon of C. butyricum coding for enzymes involved in the B12independent 1,3-propanediol pathway, and by deleting:

-   -   i) optionally, all the genes coding for the lactate        dehydrogenase (ldh) to avoid lactate production    -   ii) optionally, all the remaining genes coding for the        bi-functionnal aldehyde-alcohol dehydrogenases (adhE) to avoid        alcohol formation.

In another aspect of this invention, the flux of hydrogen production isdecreased and then the flux of reducing equivalent is redirected toward1,3-propanediol production by attenuating the gene encoding thehydrogenase (hydA).

In another aspect of the invention, the flux of 1,3-propanediolproduction is increased by introducing extra copies of the1,3-propanediol operon from C. butyricum, (coding for enzymes involvedin the B12 independent 1,3-propanediol pathway).

It is also an object of the present invention to provide a recombinantClostridum strain, useful for the process of production of1,3-propanediol at high yield.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing which is incorporated in and constitutes a partof this specification exemplifies the invention and together with thedescription, serve to explain the principles of this invention.

FIG. 1 depicts the central metabolism of different Clostridia.

1: Pyruvate-ferredoxin oxydoreductase; 2: Thiolase; 3:β-Hydroxybutyryl-CoA dehydrogenase; 4: Crotonase; 5: Butyryl-CoAdehydrogenase; 6: Lactate dehydrogenase; 7: Phospho-transacetylase; 8:Acetate kinase; 9: Acetaldehyde Ethanol deshydrogenase; 10: hydrogenase;11: CoA transférase (Acetoacetyl-CoA:acetate/butyrate:CoA transferase);12: Acetoacetate decarboxylase; 13: Phospho-transbutyrylase; 14:Butyrate kinase; 15: Butyraldehyde-Butanol dehydrogenase; 16: Glyceroldehydratase; 17: 1,3 propanediol dehydrogenase.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the following terms may be used for interpretation of theclaims and specification.

The terms “Clostridium” and “Clostridia” refer to all kind of bacteriabelonging to this family.

An appropriate culture medium refers to a culture medium optimized forthe growth and the diol production of the specifically used Clostridiumstrain.

The term “carbon substrate” or “source of carbon” means any carbonsource capable of being metabolized by a microorganism wherein thesubstrate contains at least one carbon atom. In the present inventionglycerol is the single source of carbon.

The phrase “microorganism is modified” means that the strain has beentransformed in the aim to change its genetic characteristics. Endogenousgenes can either be attenuated, deleted, or over-expressed. Exogenousgenes can be introduced, carried by a plasmid, or integrated into thegenome of the strain, to be expressed into the cell.

The term “attenuation” refers to a decreased expression of a gene or adecreased activity of the protein, product of the gene. The man skilledin the art knows numerous means to obtain this result, and for example:

-   -   Introduction of a mutation into the gene, decreasing the        expression level of this gene, or the level of activity of the        encoded protein.    -   Replacement of the natural promoter of the gene by a low        strength promoter, resulting in a lower expression.    -   Use of elements destabilizing the corresponding messenger RNA or        the protein.    -   Deletion of the gene if no expression is needed.

The term “deleted gene” means that a substantial part of the codingsequences of said gene was removed. Preferably, at least 50% of thecoding sequence was removed, and more preferably at least 80%.

The term “plasmid” or “vector” as used herein refers to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules.

In the description of the present invention, enzymes are identified bytheir specific activities. This definition thus includes allpolypeptides that have the defined specific activity also present inother organisms, more particularly in other microorganisms. Oftenenzymes with similar activities can be identified by their grouping tocertain families defined as PFAM or COG.

PFAM (protein families database of alignments and hidden Markov models;[[http://www.sanger.ac.uk/Software/Pfam/]] see world wide web atsanger.ac.uk/Software/Pfam/) represents a large collection of proteinsequence alignments. Each PFAM makes it possible to visualize multiplealignments, see protein domains, evaluate distribution among organisms,gain access to other databases, and visualize known protein structures.

COGs (clusters of orthologous groups of proteins;[[http://www.ncbi.nlm.nih.gov/COG/]] see world wide web atncbi.nlm.nih.gov/COG/) are obtained by comparing protein sequences from43 fully sequenced genomes representing 30 major phylogenic lines. EachCOG is defined from at least three lines, which permits theidentification of former conserved domains.

The means of identifying homologous sequences and their percentagehomologies are well known to those skilled in the art, and include inparticular the BLAST programs, which can be used from the website[[http://www.ncbi.nlm.nih.gov/BLAST/]] (see world wide web atncbi.nlm.nih.gov/BLAST/) with the default parameters indicated on thatwebsite. The sequences obtained can then be exploited (e.g., aligned)using, for example, the programs CLUSTALW[[(http://www.ebi.ac.uk/clustalw/)]] (see world wide web atebi.ac.uk/clustalw/) or MULTALIN[[(http://prodes.toulouse.inra.fr/multalin/cgi-bin/multalin.p1)]] (seeworld wide web at prodes.toulouse.inralemultalin/cgi-bin/multalin.p1),with the default parameters indicated on those websites.

Using the references given on GenBank for known genes, those skilled inthe art are able to determine the equivalent genes in other organisms,bacterial strains, yeasts, fungi, mammals, plants, etc. This routinework is advantageously done using consensus sequences that can bedetermined by carrying out sequence alignments with genes derived fromother microorganisms, and designing degenerate probes to clone thecorresponding gene in another organism. These routine methods ofmolecular biology are well known to those skilled in the art, and aredescribed, for example, in Sambrook et al. (1989 Molecular Cloning: aLaboratory Manual. 2^(nd) ed. Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.).

The present invention provides a method for the anaerobic production of1,3-propanediol, by culturing a Clostridium strain in an appropriateculture medium comprising glycerol as a source of carbon, wherein saidClostridium strain does not produce substantially other products of theglycerol metabolism selected among the group consisting of: butyrate,lactate, butanol and ethanol, and recovering 1,3-propanediol.

“Substantially” means that at most traces of products or reductions ofglycerol are found in the culture medium. Traces means preferablyamounts that shall not interfere with the recovery process of1,3-propanediol, more preferably less than 10 mM.

The phrase “glycerol metabolism” refers to all biochemical modificationsof glycerol happening in the bacteria. This includes the biosynthesis oforganic molecules (anabolism) and their breakdown (catabolism). Somemetabolic reactions are consuming and some others are producingNADH/NADPH. Glycerol metabolism in Clostridium strains is illustrated inFIG. 1. Intermediates as well as final products from metabolic reactionsare called metabolites.

The method of the invention is characterized by the fact that theglycerol metabolism is directed to 13,-propanediol production, and thatno other reduced products from this metabolism pathway, such asbutyrate, lactate, butanol, ethanol, are produced concomitantly with1,3-propanediol by the Clostridium. Indeed, production of these reducedproducts is consuming NADH/NADPH stock of the cell. Limiting thisconsumption will allow the reducing power to be redirected toward1,3-propanediol production.

In a specific embodiment of the invention, the 1,3-propanediol isproduced concomitantly with a single oxidized product of glycerolmetabolism, such as acetate, acetone or carbon dioxide. The term“oxidized product” refers to products produced without consumption ofthe NADH/NADPH stock of the cell

Advantageously, the Clostridium strain used in the process produces only1,3-propanediol and acetate.

According to the invention, the Clostridium strain can be modified tolimit production of metabolites from glycerol, which biosynthesispathway is NADH or NADPH consuming, except for 1,3-propanediol.

Advantageously, this modification consists of the deletion of at leastone gene coding for an enzyme involved in production of saidmetabolites.

In particular, this enzyme is involved in the production of a metaboliteselected among the group consisting of: butyrate, lactate, butanol andethanol.

In a specific embodiment of the invention, the Clostridium is naturallyproducing 1,3-propanediol, since it comprises functional endogenousgenes encoding for enzymes involved in biosynthesis of 1,3-propanediol.These genes are in particular: glycerol dehydratase and 1,3-propanedioldehydrogenase.

This strain can be genetically modified to produce 1,3-propanediol asmajor product by deleting at least one gene encoding forphospho-transbutyrylase (ptb) or butyrate kinase (buk) to block theconversion of butyryl-CoA to butyrate.

In another specific embodiment, said Clostridium said was also deletedof all the genes encoding for lactate dehydrogenase (ldh) to block theproduction of lactate.

In another specific embodiment, said Clostridium said was also deletedof all the genes encoding for bifunctionnal aldehyde-alcoholdehydrogenases (adhE) to block the production of alcohols.

Deletion of genes in Clostridia can be done using the method recentlydescribed in patent application PCT/EP2006/066997 allowing the i)replacement of the gene to delete with an erythromycin resistance geneand ii) removal of the erythromycin resistance gene by expressing theFLP recombinase.

Advantageously, the Clostridium strain is selected among the groupconsisting of C. butyricum and C. pasteurianum.

In a specific embodiment of the invention, the Clostridium strain has tobe modified to be able to produce 1,3-propanediol. The modificationconsists of the introduction of at least one heterologous gene codingfor an enzyme involved in the B-12 independent 1,3-propanediol pathway.These genes may be but are not limited to dhaB1, dhaB2, dhaT.

Advantageously, the strain is modified by introducing the operon ofClostridium butyricum coding for the enzymes involved in theB12-independent 1,3-propanediol pathway. Insertion of the operon in thechromosome can be done using the method recently described in patentapplication PCT/EP2006/066997.

In a specific embodiment of the invention, the used Clostridum strainnaturally produces butyrate but is unable to produce 1,3-propanediolprior modification; this specific Clostridium is genetically modified toproduce 1,3-propanediol by replacing at least one gene encoding for anenzyme involved in butyrate formation, in particular thephospho-transbutyrylase (ptb) or the butyrate kinase (buk), with oneheterologous gene coding for an enzyme involved in the B-12 independent1,3-propanediol pathway in the aim to:

block the conversion of butyryl-CoA to butyrate and

allow 1,3-propanediol production from glycerol in this strain.

Insertion of the operon in the chromosome and deletion of the genes canbe done using the method recently described in patent applicationPCT/EP2006/066997.

Preferentially, in this Clostridium strain, all the genes encoding forlactate dehydrogenase (ldh) are deleted to block the production oflactate.

Preferentially, in this Clostridium strain, all the genes encoding forbi-functional aldehyde-alcohol dehydrogenases (adhE) are deleted, toinhibit the production of alcohols.

Advantageously, this Clostridium strain is selected among the groupconsisting of C. acetobutylicum, C. beijerinckii, C.saccharoperbutylacetonicum C. saccharobutylicum, C. butyricum or C.cellulolyticum.

In a specific embodiment of the invention, the Clostridum naturallyproduces ethanol but is unable to produce 1,3-propanediol priormodification; this strain is genetically modified to produce1,3-propanediol by replacing at least one gene encoding forbi-functional aldehyde-alcohol dehydrogenases (adhE) with at least oneof the heterologous gene coding for an enzyme involved in the B12independent 1,3-propanediol pathway. Preferentially this heterologousgene is the operon of C. butyricum encoding for enzymes involved in theB12 independent 1,3-propanediol pathway.

This replacement leads to:

a decrease of the conversion of acetyl-CoA to ethanol, and

1,3-propanediol production from glycerol.

Preferably, in this Clostridium strain, all the genes encoding forlactate dehydrogenase (ldh) are deleted to inhibit the production oflactate.

Preferably, in this Clostridium strain, all the remaining genes encodingfor bi-functional aldehyde-alcohol dehydrogenases (adhE) are deleted toblock the production of alcohols.

Insertion of the operon in the Clostridium chromosome and deletion ofthe previously cited genes can be done using the method recentlydescribed in patent application PCT/EP2006/066997.

Advantageously, this Clostridium strain is selected among the groupconsisting of Clostridium thermocellum, Clostridium saccharolyticum (nowThermoanaerobacter saccharolyticum), Clostridium thermosulfurogenes (nowThermoanaerobacter thermosulfurigenes) or Clostridiumthermohydrosulfuricum (now Thermoanaerobacter ethanolicus).

In a specific embodiment of the invention, the Clostridium strain has adecreased flux of hydrogen production and consequently presents aredirection of the flux of reducing equivalent toward 1,3-propanediolproduction. This result may be achieved by various means, and inparticular by attenuating the gene encoding the hydrogenase (hydA), anenzyme that provides a sink for reducing equivalent in the form ofhydrogen production. Attenuation of hydA can be done by replacing thenatural promoter by a low strength promoter or by using an elementdestabilizing the corresponding messenger RNA or the protein. If needed,complete attenuation of the gene can also be achieved by partial orcomplete deletion of the corresponding DNA sequence.

In another embodiment of the invention, the used Clostridium strainpresents an increased flux of 1,3-propanediol production; this result isachieved by introducing extra copies of the 1,3-propanediol operon fromC. butyricum, (coding for enzymes involved in the B12 independent1,3-propanediol pathway) either over-expressed by a plasmid orintegrated into the chromosome of the recombinant Clostridium. Forexample the pSPD5 plasmid can be used for an over-expression of the1,3-propanediol operon.

In another aspect of the invention, the Clostridium strain is modifiedto be able to convert acetate to acetone. This modification can beobtained by introducing into the microorganism an artificial “acetoneoperon” containing the thl, ctfAB and adc genes coding respectively forthe thiolase, the CoA-transferase and the aceto-acetate decarboxylase,these three enzymes being involved in acetone formation in C.acetobutylicum and C. beijerinckii. This artificial operon can be eithercarried by a plasmid or can be integrated into the chromosome of thetransformed Clostridium.

In another embodiment, the invention provides a method for thefermentative preparation of 1,3-propanediol at high yield, comprising:

-   -   (a) contacting a Clostridium strain with glycerol for a        fermentation process whereby 1,3-propanediol is produced,    -   (b) isolating 1,3-propanediol and optionally a single oxidized        product of the glycerol metabolism (mainly acetate or acetone)        by distillation.

The fermentation is generally conducted in fermentors with an inorganicculture medium of known defined composition adapted to the bacteriaused, containing at least glycerol, and if necessary a co-substratenecessary for the production of the metabolite.

This process can be realized in a batch process as well as in acontinuous process. The man skilled in the art knows how to manage eachof these experimental conditions, and to define the culture conditionsfor the microorganisms according to the invention. In particular theclostridia are fermented at a temperature between 20° C. and 60° C.,preferentially between 25° C. and 40° C. for mesophilic clostridia andbetween 45 and 60° C. for thermophilic Clostridia.

The invention is also related to the microorganism as describedpreviously. Preferably, this microorganism is selected among the groupconsisting of C. butyricum, C. pasteurianum, C. acetobutylicum, C.beijerinckii, C. saccharoperbutylacetonicum C. saccharobutylicum, C.butyricum, C. cellulolyticum, Clostridium thermocellum, Clostridiumsaccharolyticum (now Thermoanaerobacter saccharolyticum), Clostridiumthermosulfurogenes (now Thermoanaerobacter thermosulfurigenes) orClostridium thermohydrosulfuricum (now Thermoanaerobacter ethanolicus).

EXAMPLE 1 Construction of a Recombinant Clostridium AcetobutylicumProducing 1,3-Propanediol and Unable to Produce Butyrate and Butanol: C.Acetobutylicum ΔpSOL1 Δcac1515 Δupp Δbuk::PDO

To obtain a strain that can be genetically manipulated and is unable toproduce butanol and acetone, we first cure the pSOL1 megaplasmid fromthe C. acetobutylicum Δcac1515 Δupp strain (described in patentapplication PCT/EP2006/066997) by i) running 20 sub-cultures in glucoseMS medium and ii) by selection on agar plates (containing starch (2%)and glucose (0.2%) as described by Sabathe et al (2003)) of clonesproducing small hallo of starch hydrolysis to identify a C.acetobutylicum ΔpSOL1 Δcac1515 Δupp strain. To delete the buk gene andintroduce the 1,3-propanediol operon from C. butyricum, the homologousrecombination strategy described by Croux & Soucaille (2006) in patentapplication PCT/EP2006/066997 is used. A buk deletion cassetteintegrating the 1,3-propanediol operon from C. butyricum in thepCons::upp vector was constructed as follows.

Two DNA fragments surrounding buk were PCR amplified with the Pwopolymerase with total DNA from C. acetobutylicum as template and twospecific couples of olignonucleotides. With the couples of primers BUK1-BUK 21 and BUK 31-BUK 4, two DNA fragments were respectively obtained.Both primers BUK 1 and BUK 4 introduce a BamHI site while primers BUK 21and BUK 31 have a complementary region which introduces pvuII and NruIsites. DNA fragments BUK 1-BUK 21 and BUK 31-BUK 4 were joined in a PCRfusion experiment with primers BUK 1 and BUK 4 and the resultingfragment was cloned in pCR4-TOPO-Blunt to yield pTOPO:buk. At the uniquenruI site of pTOPO:buk, an antibiotic resistance MLS gene with FRTsequences on both sides was introduced from the 1372 bp StuI fragment ofpUC18-FRT-MLS2. The BUK deletion cassette obtained after BamHI digestionof the resulting plasmid was cloned into pCons::upp at the BamHI site toyield the pREPΔBUK::upp plasmid. At the unique pvuII site ofpREPΔBUK::upp, the 1,3-propanediol operon was introduced as a 4854 bpblunt end Klenow treated SalI fragment of pSPD5 plasmid.

The pREPΔBUK::PDO::upp plasmid was used to transform by electroporationC. acetobutylicum ΔpSOL1 Δcac15Δupp strain. After selection on Petriplate for clones resistant to erythromycin (40 μg/ml), one colony wascultured for 24 hours in Glycerol liquid synthetic medium witherythromycin at 40 μg/ml and 100 μl of undiluted culture was plated onRCGA (Reinforced Clostridium medium where starch and glucose arereplaced by glycerol as a carbon source) with erythromycin at 40 μg/mland 5-FU at 400 μM. Colonies resistant to both erythromycin and 5-FUwere replica plated on both RCA with erythromycin at 40 μg/ml and RCAwith thiamphenicol at 50 μg/ml to select clones where 5-FU resistance isalso associated with thiamphenicol sensitivity. The genotype of clonesresistant to erythromycin and sensitive to thiamphenicol was checked byPCR analysis (with primers BUK 0 and BUK 5 located outside of the bukdeletion cassette).

The ΔpSOL1Δcac15ΔuppΔbuk::PDO::mls^(R) strain which have lostpREPΔbuk::upp was isolated.

The ΔpSOL1Δcac15ΔuppΔbuk::PDO::mls^(R) strain was transformed withpCLF1.1 vector expressing the Flp1 gene encoding the Flp recombinasefrom S. cerevisiae. After transformation and selection for resistance tothiamphenicol (50 μg/ml) on Petri plate, one colony was cultured onsynthetic liquid medium with thiamphenicol at 50 μg/ml and appropriatedilutions were plated on RCA with thiamphenicol at 50 μg/ml.Thiamphenicol resistant clones were replica plated on both RCA witherythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. Thegenotype of clones with erythromycin sensitivity and thiamphenicolresistance was checked by PCR analysis with primers BUK 0 and BUK 5. Twosuccessive 24 hours cultures of the Δcac15ΔuppΔbuk strain witherythromycin sensitivity and thiamphenicol resistance were carried outin order to lose pCLF1.1. The ΔpSOL1Δcac15ΔuppΔbuk::PDO strain which haslost pCLF1.1 was isolated according to its sensitivity to botherythromycin and thiamphenicol.

TABLE 1 Name Primer sequences Buk 1 aaaa

tagtaaaagggagtgtacgaccagtg Buk 21

gattattagtaatctatacatgttaacattcctccac Buk 31

acttcttgcacttgcagaaggtggac Buk 4 aaaa

tctaaattctgcaatatatgccccccc Buk 0 ataacaggatatatgctctctgacgcgg Buk 5gatcatcactcattttaaacatggggcc

EXAMPLE 2 Construction of Strains unable to Produce Butyrate, Acetoneand Lactate: C. Acetobutylicum ΔpSOL1 Δcac1515 Δupp Δbuk::PDO Δldh

To delete the ldh gene, the homologous recombination strategy describedby Croux & Soucaille (2006) in patent application PCT/EP2006/066997 isused. This strategy allows the insertion of an erythromycin resistancecassette, while deleting most of the genes concerned. The ldh deletioncassette in pCons::upp was constructed as follows.

Two DNA fragments surrounding ldh (CAC267) were PCR amplified with thePwo polymerase with total DNA from C. acetobutylicum as template and twospecific couples of olignonucleotides. With the couples of primers LDH1-LDH 2 and LDH 3-LDH 4, 1135 bp and 1177 bp DNA fragments wererespectively obtained. Both primers LDH 1 and LDH 4 introduce a BamHIsite while primers LDH 2 and LDH 3 have a complementary region whichintroduces a StuI site. DNA fragments LDH 1-LDH 2 and LDH 3-LDH 4 werejoined in a PCR fusion experiment with primers LDH 1 and LDH 4 and theresulting fragment was cloned in pCR4-TOPO-Blunt to yield pTOPO:LDH. Atthe unique StuI site of pTOPO:LDH, an antibiotic resistance MLS genewith FRT sequences on both sides was introduced from the 1372 bp StuIfragment of pUC18-FRT-MLS2. The UPP deletion cassette obtained afterBamHI digestion of the resulting plasmid was cloned into pCons::upp atthe BamHI site to yield the pREPΔLDH::upp plasmid.

The pREPALDH::upp plasmid was used to transform by electroporation C.acetobutylicum ΔpSOL1Δcac15ΔuppΔbuk::PDO strain. After selection onPetri plate (on RCGA) for clones resistant to erythromycin (40 μg/ml),one colony was cultured for 24 hours in liquid glycerol synthetic mediumwith erythromycin at 40 μg/ml and 100 μl of undiluted culture was platedon RCGA with erythromycin at 40 μg/ml and 5-FU at 400 μM. Coloniesresistant to both erythromycin and 5-FU were replica plated on both RGCAwith erythromycin at 40 μg/ml and RGCA with thiamphenicol at 50 μg/ml toselect clones where 5-FU resistance is also associated withthiamphenicol sensitivity. The genotype of clones resistant toerythromycin and sensitive to thiamphenicol was checked by PCR analysis(with primers LDH 0 and LDH 5 located outside of the ldh deletioncassette). The ΔΔpSOL1Δcac15ΔuppΔbuk::PDOΔldh::mls^(R) strain which havelost pREPΔLDH::upp was isolated.

The ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldh::mls^(R) strain was transformed withpCLF1.1 vector expressing the Flp1 gene encoding the Flp recombinasefrom S. cerevisiae. After transformation and selection for resistance tothiamphenicol (50 μg/ml) on Petri plate, one colony was cultured onsynthetic liquid medium with thiamphenicol at 50 μg/ml and appropriatedilutions were plated on RCA with thiamphenicol at 50 μg/ml.Thiamphenicol resistant clones were replica plated on both RCA witherythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. Thegenotype of clones with erythromycin sensitivity and thiamphenicolresistance was checked by PCR analysis with primers LDH 0 and LDH 5. Twosuccessive 24 hours cultures of the ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldh strainwith erythromycin sensitivity and thiamphenicol resistance were carriedout in order to lose pCLF1.1. The ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldh strainwhich has lost pCLF1.1 was isolated according to its sensitivity to botherythromycin and thiamphenicol.

TABLE 2 Name Primer sequences Ldh 1AAAAGGATCCGCTTTAAAATTTGGAAAGAGGAAGTTGTG Ldh 2GGGGAGGCCTAAAAAGGGGGTTAGAAATCTTTAAAAATTTCTCTATAGAGCCCATC Ldh 3CCCCCTTTTTAGGCCTCCCCGGTAAAAGACCTAAACTCCAAGGGTGGAGGCTAGGTC Ldh 4AAAAGGATCCCCCATTGTGGAGAATATTCCAAAGAAGAAAATAATTGC Ldh 0CAGAAGGCAAGAATGTATTAAGCGGAAATGC Ldh 5 CTTCCCATTATAGCTCTTATTCACATTAAGC

EXAMPLE 3 Construction of Strains with Lower Hydrogen Production: C.Acetobutylicum ΔpSOL1 Δcac1515 Δupp Δbuk::PDO Δldh ΔhydA

To delete the hydA gene, the homologous recombination strategy describedby Croux & Soucaille (2006) in patent application PCT/EP2006/066997 isused. This strategy allows the insertion of an erythromycin resistancecassette, while deleting most of the genes concerned. The hydA deletioncassette in pCons::upp was constructed as follows. Two DNA fragmentssurrounding hydA (CACO28) were PCR amplified with the Pwo polymerasewith total DNA from C. acetobutylicum as template and two specificcouples of olignonucleotides. With the couples of primers HYD 1-HYD 2and HYD 3-HYD 4, 1269 bp and 1317 bp DNA fragments were respectivelyobtained. Both primers HYD 1 and HYD 4 introduce a BamHI site whileprimers HYD 2 and HYD 3 have a complementary region which introduces aStuI site. DNA fragments HYD 1-HYD 2 and HYD 3-HYD 4 were joined in aPCR fusion experiment with primers HYD 1 and HYD 4 and the resultingfragment was cloned in pCR4-TOPO-Blunt to yield pTOPO:HYD. At the uniqueStuI site of pTOPO:HYD, an antibiotic resistance MLS gene with FRTsequences on both sides was introduced from the 1372 bp StuI fragment ofpUC18-FRT-MLS2. The UPP deletion cassette obtained after BamHI digestionof the resulting plasmid was cloned into pCons::upp at the BamHI site toyield the pREPΔHYD::upp plasmid.

The pREPΔHYD::upp plasmid was used to transform by electroporation C.acetobutylicum ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldh strain. After selection onPetri plate (RCGA) for clones resistant to erythromycin (40 μg/ml), onecolony was cultured for 24 hours in glycerol liquid synthetic mediumwith erythromycin at 40 μg/ml and 100 μl of undiluted culture was platedon RCA with erythromycin at 40 μg/ml and 5-FU at 400 μM. Coloniesresistant to both erythromycin and 5-FU were replica plated on both RCGAwith erythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml toselect clones where 5-FU resistance is also associated withthiamphenicol sensitivity. The genotype of clones resistant toerythromycin and sensitive to thiamphenicol was checked by PCR analysis(with primers HYD 0 and HYD 5 located outside of the hydA deletioncassette). The ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldhΔhydA::mls^(R) strain whichhave lost pREPΔHYD::upp was isolated.

The ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldhΔhydA::mls^(R) strain was transformedwith pCLF1.1 vector expressing the Flp1 gene encoding the Flprecombinase from S. cerevisiae. After transformation and selection forresistance to thiamphenicol (50 μg/ml) on Petri plate, one colony wascultured on synthetic liquid medium with thiamphenicol at 50 μg/ml andappropriate dilutions were plated on RCA with thiamphenicol at 50 μg/ml.Thiamphenicol resistant clones were replica plated on both RCA witherythromycin at 40 μg/ml and RCA with thiamphenicol at 50 μg/ml. Thegenotype of clones with erythromycin sensitivity and thiamphenicolresistance was checked by PCR analysis with primers HYD 0 and HYD 5. Twosuccessive 24 hours cultures of the ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldhΔhydAstrain with erythromycin sensitivity and thiamphenicol resistance werecarried out in order to lose pCLF1.1. TheΔpSOL1Δcac15ΔuppΔbuk::PDOΔldhΔhydA strain which has lost pCLF1.1 wasisolated according to its sensitivity to both erythromycin andthiamphenicol.

TABLE 3 Name Primer sequences Hyd 1 AAAA

GCCTCTTCTGTATTATGCAAGGAAAGCAGCTGC Hyd 2GGGGAGGCCTAAAAAGGGGGTATATAAAATAAATGTGCCTTAACATCTAA GTTGAGGCC Hyd 3CCCCCTTTTTAGGCCTCCCCGTTTATCCTCCCAAAATGTAAAATATAATTAAAATATATTAATAAACTTCGATTAATAAACTTCG Hyd 4AAAAGGATCCCCTTTTAGCGTATAAAGTTTTATATAGCTATTG Hyd 0CATGTTCTATTGTTACTATGGAAGAGGTAGTAG Hyd 5 GCAGTTATTATAAATGCTGCTACTAGAGC

EXAMPLE 4 Construction of Strains with Increase Flux in the1,3-Propanediol Pathway: C. acetobutylicum ΔpSOL1 Δcac1515 ΔuppΔbuk::PDO Δldh pSPD5

To construct a strain that converts glycerol to 1,3-propanediol andacetate at higher flux we introduce the pSPD5 plasmid (described inpatent application WO01/04324) that expressed as an operon the B12independent 1,3-propanediol pathway from C. butyricum. The pSPD5 plasmidwas used to transform by electroporation of C. acetobutylicumΔpSOL1Δcac15ΔuppΔbuk::PDOΔldh strain. After selection on Petri plate(RCGA) for clones resistant to erythromycin (40 μg/ml), one colony wascultured for 24 hours in glycerol liquid synthetic medium witherythromycin at 40 μg/ml and use to extract the pSPD5 plasmid that wascharacterized by its restriction profile.

EXAMPLE 5 Construction of Strains Producing 1,3-propanediol and Acetone:C. Acetobutylicum ΔpSOL1 Δcac1515 Δupp Δbuk::PDO Δldh pSOS95 thl

To construct a strain that converts acetate to acetone the pSOS 95 thlplasmid expressing a synthetic acetone operon was constructed. For thispurpose the thl gene encoding thiolase (from C. acetobutylicum) wasintroduced at the BamHI site of the pSOS95 vector (genbank accession n°AY187686) already expressing as a synthetic operon the ctfAB and adcgenes. The thl gene was PCR amplified with the Pwo polymerase with totalDNA from C. acetobutylicum as template and two specific couples ofolignonucleotides. With the couple of primers THL 1-THL2 a 1.2 kbp DNAfragment was obtained and digested with BamHI and Bg1II, two restrictionsites that were respectively introduced by primers THL 1 and THL2. Afterligation to the BamhI digested pSOS95, the pSOS95-thl plasmid wasobtained.

This pSOS95-thl plasmid was used to transform by electroporation C.acetobutylicum ΔpSOL1Δcac15ΔuppΔbuk::PDOΔldh strain. After selection onPetri plate (RCGA) for clones resistant to erythromycin (40 μg/ml), onecolony was cultured for 24 hours in glycerol liquid synthetic mediumwith erythromycin at 40 μg/ml and use to extract the pSOS95-thl plasmidthat was characterized by its restriction profile.

TABLE 4 Name Primer sequences THL 1 cgc

tttatctgttaccccgtatcaaaatttagg THL 2 ga

TCTAGCACTTTTCTAGCAATATTGC

EXAMPLE 6 Batch Fermentation of 1,3-Propanediol Producing Strains

Strains were initially analyzed in anaerobic flask cultures in thesynthetic medium described by Soni et al (Soni et al, 1986, Appl.Microbiol. Biotechnol. 32:120-128) supplemented with 2.5 g/1 of ammoniumacetate and with replacement of glucose by glycerol. An overnightculture at 35° C. was used to inoculate a 30 ml culture to an OD600 of0.05. After incubation of the culture for 3 days at 35° C., glycerol,organic acids and 1,3-propanediol were analyzed by HPLC using a BioradHPX 97H column for the separation and a refractometer for the detection.

Strains with the correct phenotype were subsequently tested underproduction conditions in 300 ml fermentors (DASGIP) using an anaerobicbatch protocol.

For this purpose the fermentor was filled with 250 ml of syntheticmedium, sparged with nitrogen for 30 min and inoculated with 25 ml ofpreculture to an optical density (OD600nm) between 0.05 and 0.1.

The temperature of the culture was maintained constant at 35° C. and thepH was permanently adjusted at 6.5 using an NH₄OH solution. Theagitation rate was maintained at 300 rpm during the fermentation.

EXAMPLE 7 Continuous Fermentation of 1,3-Propanediol and AcetateProducing Strains

The best 1,3-propanediol and acetate producing strain was analyzed inchemostat cultures in the synthetic medium described by Soni et al (Soniet al, 1987, Appl. Microbiol. Biotechnol.) except that glucose wasreplaced by glycerol. An overnight culture at 35° C. was used toinoculate a 300 ml fermentors (DASGIP) using an anaerobic chemostatprotocol.

For this purpose the fermentor was filled with 250 ml of syntheticmedium, sparged with nitrogen for 30 min and inoculated with 25 ml ofpreculture to an optical density (OD600nm) between 0.05 and 0.1. After12 hours of batch culture at 35° C., pH 6.5 (regulated using an NH₄OHsolution) and an agitation rate of 300 rpm, the fermentor wascontinuously fed with oxygen free synthetic medium at a dilution rate of0.05 h-1 while the volume was kept constant by sequential removal offermentated medium. Stability of the culture was followed by productsanalysis using the HPLC protocol previously described.

REFERENCES

-   Gonzalez-Pajuelo M, Meynial-Salles I, Mendes F, Andrade J C,    Vasconcelos I, Soucaille P.-   Metabolic engineering of Clostridium acetobutylicum for the    industrial production of 1,3-propanediol from glycerol.-   Metab Eng. 2005; 7:329-36.-   Green E M, Boynton Z L, Harris L M, Rudolph F B, Papoutsakis E T,    Bennett G N.-   Genetic manipulation of acid formation pathways by gene inactivation    in Clostridium acetobutylicum ATCC 824.-   Microbiology. 1996, 142:2079-86.-   Soni B. K., Soucaille P. Goma G.-   Continuous acetone butanol fermentation: influence of vitamins on    the metabolic activity of Clostridium acetobutylicum.-   Appl. Microbiol. Biotechnol. 1987. 27:1-5.

1. A method for the anaerobic production of 1,3-propanediol comprising:culturing a Clostridium strain in an appropriate culture mediumcomprising glycerol as a source of carbon; and recovering1,3-propanediol, wherein said Clostridium strain does not producesubstantially other products of the glycerol metabolism selected from:butyrate, lactate, butanol and ethanol; wherein the Clostridium strainproduces only 1,3-propanediol and acetate from glycerol; wherein theClostridium strain comprises functional endogenous genes for productionof 1,3-propanediol; and wherein all the ldh genes coding for lactatedehydrogenases are deleted.
 2. A method for the anaerobic production of1,3-propanediol comprising: culturing a Clostridium strain in anappropriate culture medium comprising glycerol as a source of carbon;and recovering 1,3-propanediol, wherein said Clostridium strain does notproduce substantially other products of the glycerol metabolism selectedfrom: butyrate, lactate, butanol and ethanol; wherein the Clostridiumstrain produces only 1,3-propanediol and acetate from glycerol; whereinthe Clostridium strain comprises functional endogenous genes forproduction of 1,3-propanediol; and wherein all the adhE genes coding foraldehyde-alcohol dehydrogenases are deleted.
 3. The method according toclaim 1 or 2 wherein the Clostridium strain is selected from C.butyricum and C. pasteurianum.
 4. A method for the anaerobic productionof 1,3-propanediol comprising: culturing a Clostridium strain in anappropriate culture medium comprising glycerol as a source of carbon;and recovering 1,3-propanediol; wherein said Clostridium strain does notproduce substantially other products of the glycerol metabolism selectedfrom: butyrate, lactate, butanol and ethanol; wherein the Clostridiumstrain produces only 1,3-propanediol and acetate from glycerol; whereinthe Clostridium strain is modified to produce 1,3-propanediol byintroducing at least one heterologous gene coding for an enzyme involvedin the B-12 independent 1,3-propanediol pathway; wherein the Clostridiumstrain, prior to modification, can produce butyrate and the at least oneheterologous gene is introduced to replace at least one gene coding foran enzyme involved in butyrate formation; wherein the gene coding for anenzyme involved in butyrate formation is ptb encodingphospho-transbutyrylase or buk encoding butyrate kinase; and wherein allthe ldh genes coding for lactate dehydrogenases are deleted.
 5. A methodfor the anaerobic production of 1,3-propanediol comprising: culturing aClostridium strain in an appropriate culture medium comprising glycerolas a source of carbon; and recovering 1,3-propanediol; wherein saidClostridium strain does not produce substantially other products of theglycerol metabolism selected from: butyrate, lactate, butanol andethanol; wherein the Clostridium strain produces only 1,3-propanedioland acetate from glycerol; wherein the Clostridium strain is modified toproduce 1,3-propanediol by introducing at least one heterologous genecoding for an enzyme involved in the B-12 independent 1,3-propanediolpathway; wherein the Clostridium strain, prior to modification, canproduce butyrate and the at least one heterologous gene is introduced toreplace at least one gene coding for an enzyme involved in butyrateformation; and wherein all the adhE genes coding for aldehyde-alcoholdehydrogenases are deleted.
 6. The method according to claim 4 or 5wherein the Clostridium strain is selected from C. acetobutylicum, C.beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, C.butyricum and C. cellulolyticum.
 7. A method for the anaerobicproduction of 1,3-propanediol comprising: culturing a Clostridium strainin an appropriate culture medium comprising glycerol as a source ofcarbon; and recovering 1,3-propanediol; wherein said Clostridium straindoes not produce substantially other products of the glycerol metabolismselected from: butyrate, lactate, butanol and ethanol; wherein theClostridium strain produces only 1,3-propanediol and acetate fromglycerol; wherein the Clostridium strain is modified to produce1,3-propanediol by introducing at least one heterologous gene coding foran enzyme involved in the B-12 independent 1,3-propanediol pathway; andwherein the Clostridium strain, prior to modification, can produceethanol and the at least one heterologous gene is introduced to replaceat least one gene coding for an enzyme involved in ethanol formation. 8.The method of claim 7, wherein the gene coding for an enzyme involved inethanol formation is selected among the adhE genes coding foraldehyde-alcohol dehydrogenases.
 9. The method according to claim 8wherein all the ldh genes coding for lactate dehydrogenases are deleted.10. The method according to claim 7 wherein all the remaining adhE genescoding for aldehyde-alcohol dehydrogenases are deleted.
 11. The methodaccording to claim 7 wherein the Clostridium strain is selected fromClostridium thermocellum, Clostridium saccharolyticum (nowThermoanaerobacter saccharolyticum), Clostridium thermosulfurogenes (nowThermoanaerobacter thermosulfurigenes) and Clostridiumthermohydrosulfuricum (now Thermoanaerobacter ethanolicus).
 12. A methodfor the anaerobic production of 1,3-propanediol comprising: culturing aClostridium strain in an appropriate culture medium comprising glycerolas a source of carbon; and recovering 1,3-propanediol; wherein saidClostridium strain does not produce substantially other products of theglycerol metabolism selected from: butyrate, lactate, butanol andethanol; wherein the Clostridium strain produces only 1,3-propanedioland acetate from glycerol; wherein the microorganism is modified todecrease the hydrogen flux and to re-direct the reducing power to1,3-propanediol production; and wherein the hydA gene involved in thehydrogen flux is attenuated.
 13. A method for the anaerobic productionof 1,3-propanediol comprising: culturing a Clostridium strain in anappropriate culture medium comprising glycerol as a source of carbon;and recovering 1,3-propanediol; wherein said Clostridium strain does notproduce substantially other products of the glycerol metabolism selectedfrom: butyrate, lactate, butanol and ethanol; wherein the Clostridiumstrain produces only 1,3-propanediol and acetate from glycerol; andwherein the microorganism is modified to convert acetate to acetone. 14.The method as claimed in claim 13 wherein the genes coding for theenzymes involved in acetone formation are exogenous and are introducedinto the Clostridium strain.
 15. A 1,3-propanediol-producing Clostridiumstrain, wherein the strain does not produce substantially other productsof the glycerol metabolism chosen from butyrate, lactate, butanol, andethanol, wherein the strain comprises functional endogenous genes forproduction of 1,3-propanediol, and wherein all the ldh genes coding forlactate dehydrogenases are deleted.
 16. A 1,3-propanediol-producingClostridium strain, wherein the strain does not produce substantiallyother products of the glycerol metabolism chosen from butyrate, lactate,butanol, and ethanol, wherein the strain comprises functional endogenousgenes for production of 1,3-propanediol, and wherein all the adhE genescoding for aldehyde-alcohol dehydrogenases are deleted.
 17. TheClostridium strain of claim 15 or claim 16, wherein the strain is C.butyricum or C. pasteurianum.
 18. A 1,3-propanediol-producingClostridium strain, wherein the strain does not produce substantiallyother products of the glycerol metabolism chosen from butyrate, lactate,butanol, and ethanol; wherein the strain is modified to produce1,3-propanediol by introducing at least one heterologous gene coding foran enzyme involved in the B-12 independent 1,3-propanediol pathway;wherein the strain, prior to modification, can produce butyrate and theat least one heterologous gene is introduced to replace at least onegene coding for an enzyme involved in butyrate formation; wherein thegene coding for an enzyme involved in butyrate formation is ptb encodingphospho-transbutyrylase or buk encoding butyrate kinase; and wherein allthe ldh genes coding for lactate dehydrogenases are deleted.
 19. A1,3-propanediol-producing Clostridium strain, wherein the strain doesnot produce substantially other products of the glycerol metabolismchosen from butyrate, lactate, butanol, and ethanol; wherein the strainis modified to produce 1,3-propanediol by introducing at least oneheterologous gene coding for an enzyme involved in the B-12 independent1,3-propanediol pathway; wherein the strain, prior to modification, canproduce butyrate and the at least one heterologous gene is introduced toreplace at least one gene coding for an enzyme involved in butyrateformation; and wherein all the adhE genes coding for aldehyde-alcoholdehydrogenases are deleted.
 20. The Clostridium strain of claim 18 or19, wherein the strain is C. acetobutylicum, C. beijerinckii, C.saccharoperbutylacetonicum, C. saccharobutylicum, C. butyricum, or C.cellulolyticum.
 21. A 1,3-propanediol-producing Clostridium strain,wherein the strain does not produce substantially other products of theglycerol metabolism chosen from butyrate, lactate, butanol, and ethanol;wherein the strain is modified to produce 1,3-propanediol by introducingat least one heterologous gene coding for an enzyme involved in the B-12independent 1,3-propanediol pathway; and wherein the strain, prior tomodification, can produce ethanol and the at least one heterologous geneis introduced to replace at least one gene coding for an enzyme involvedin ethanol formation.
 22. The Clostridium strain of claim 21, whereinthe gene coding for an enzyme involved in ethanol formation is chosenfrom the adhE genes coding for aldehyde-alcohol dehydrogenases.
 23. TheClostridium strain of claim 22, wherein all the ldh genes coding forlactate dehydrogenases are deleted.
 24. The Clostridium strain of claim21, wherein all the remaining adhE genes coding for aldehyde-alcoholdehydrogenases are deleted.
 25. The Clostridium strain of claim 21,wherein the strain is Clostridium thermocellum, Clostridiumsaccharolyticum (now Thermoanaerobacter saccharolyticum), Clostridiumthermosulfurogenes (now Thermoanaerobacter thermosulfurigenes), orClostridium thermohydrosulfuricum (now Thermoanaerobacter ethanolicus).26. A 1,3-propanediol-producing Clostridium strain, wherein the straindoes not produce substantially other products of the glycerol metabolismchosen from butyrate, lactate, butanol, and ethanol; wherein the strainis modified to produce 1,3-propanediol by introducing at least oneheterologous gene coding for an enzyme involved in the B-12 independent1,3-propanediol pathway; wherein the microorganism is modified todecrease the hydrogen flux and to redirect the reducing power to1,3-propanediol production; and wherein the hydA gene involved inhydrogen flux is attenuated.
 27. A 1,3-propanediol-producing Clostridiumstrain, wherein the strain does not produce substantially other productsof the glycerol metabolism chosen from butyrate, lactate, butanol, andethanol; wherein the strain is modified to produce 1,3-propanediol byintroducing at least one heterologous gene coding for an enzyme involvedin the B-12 independent 1,3-propanediol pathway; and wherein themicroorganism is modified to convert acetate to acetone.
 28. TheClostridium strain of claim 27, wherein the genes coding for the enzymesinvolved in acetone formation are exogenous and are introduced into theClostridium strain.